Evidence for local platelet activation ... patients with pulmonary hypertension secondary ...

Eur Aeaplr J
1991, 4, 147-151
Evidence for local platelet activation in pulmonary vessels in
patients with pulmonary hypertension secondary to chronic
obstructive pulmonary disease
C. Rostagno, D. Prisco, M. Boddi, L. Poggesi
Evidence for local platelet activation in pulmonary vessels in patients with
pulmonary hypertension secondary to chronic obstructive pulmonary disease.
C. Rostagno, D. Prisco, M. Boddi, L. Poggesi.
ABSTRACT: To Investigate the relationships between local platelet
activation In pulmonary vessels and pulmonary artery pressure circulating
platelet aggregates and plasma beta-thromboglobulin (beta-TG) levels were
evaluated In peripheral venous blood and blood from different sites of
pulmonary circulation (right ventricle, pulmonary artery and arteriolocapillary bed) in 29 patients with COPD. Fifteen had pulmonary
hypertension and 14 normal pulmonary artery pressure. In normotensive
COPD no significant differences could be found in platelet aggregation and
beta-TG levels among different sampling sites. On the contrary in patients
with pulmonary hypertension a significant Increase of platelet aggregates
and beta-TG levels was found in blood withdrawn from the arteriolocapillary bed. A significant correlation was demonstrated between platelet
aggregation and both mean pulmonary artery pressure and pulmonary
vascular resistance. These results indicate that in patients with pulmonary
hypertension secondary to COPD a local platelet activation in pulmonary
vessels does occur and may contribute to the maintainance of elevated
pulmonary vascular resistance.
Clinica Medica I, University of Florence, Italy.
Correspondence: C. Rostagno, Clinica Medica I,
University of Florence, Viale Morgagni 85, 1-50137
Firenze, Italy.
Keywords: Chronic obstructive pulmonary disease,
platelets; pulmonary hypertension.
Received: February 16, 1990; accepted after revision
October 17, 1990.
Eur Respir J., 1991, 4, 147-151.
The high prevalence of thrombotic lesions in
pulmonary arteries from patients affected by pulmonary
hypertension due to chronic obstructive pulmonary
disease (COPD) [1, 2] has focused the attention of
investigators on a possible role for platelets in the
pathogenesis and/or maintainance of increased pulmonary
vascular resistance in these patients. A shortened platelet
survival time due to increased consumption was shown
in patients with COPD [3]. Furthermore, increased
plasma levels of beta-thromboglobulin (beta-TG), an index
of platelet activation, and enhanced platelet aggregation
have been reported in peripheral venous blood in patients
with pulmonary hypertension due to COPD [4-6].
Although these findings indicate a relationship between
increase in pulmonary vascular resistance and platelet
activation they are not conclusive per se. Indeed platelet
activation in patients with COPD could actually occur in
systemic circulation as a result of hypoxaemia, acidosis
or hyperviscosity, all characteristic findings in chronic
lung disease. Thus, we planned this study in order to
investigate whether platelet activation may play a role in
the occurrence of pulmonary hypertension in patients with
COPD.
Patients and methods
Twenty nine patients (26 men and 3 women, mean age
60.2 yrs) with chronic outflow obstruction underwent right
cardiac catheterization for diagnostic purposes. Diagnosis
was established on the basis of clinical hystory, physical
examination, chest X-ray and physiological ventilation
tests. Characteristics of patients are reported in table 1
and 2. Fifteen of these patients had mean pulmonary
artery pressure exceeding 20 mmHg (mean 33.43±11.22
mmHg) and pulmonary vascular resistance exceeding 160
dynes·s·cm-5 (mean 320.9±52.4 dynes·s·cm-5), while the
other 14 had normal mean pulmonary artery pressure
(mean 13.9±3.6 mmHg) and pulmonary vascular
resistance (mean 113±52.4 dynes·s·cm- 5). Clinical
conditions were stable in all patients in the two months
before examination and no patient was on antiplatelet
treatment in the two weeks preceding the study. Blood
samples for beta-TG and platelet aggregate assays were
collected from right ventricle, pulmonary artery and
pulmonary capillaries and from an antecubital vein.
Venous samples were also collected from 15 healthy
volunteers (control group) of equivalent age and sex.
148
C. ROSTAGNO ET AL.
Table 1.- Patient characteristics
Sex
Group I
1 F.A.
2 M.B.L.
3 A.G.
4 A.T.
5 A.G.
6 L.V.
7 G.C.
8 A.L.
9 A.C.
10 P.G.
11 F.G.
12 G.D.
13 L.P.
14 V.D.
Groupll
1 R.B.
2 P.P.
3 F.P.
4 M.S.
5 R.B.
6 A.S.
7 L.G.
8 G.C.
9 G.C.
10 M.V.
11 E.G.
12 P.D.
13 B.M.
14 D.M.
15 G.F.
M
F
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
F
M
M
M
M
F
M
M
M
M
Age
yrs
48
30
67
64
76
70
65
68
73
74
57
72
68
71
72
45
50
62
64
60
62
54
36
30
50
60
58
69
62
g·[·l
Ht
%
x109·[·1
CB
Asthma
Emphys
CB
COPD
CB
CB
Emphys
COPD
COPD
Asthma
CB
CB
CB
156
146
157
154
158
143
149
146
141
143
145
152
147
165
49
42
46
45
48
42
44
42
43
41
42
46
43
49
225
186
300
234
254
218
255
278
236
248
257
197
218
310
2.72
2.20
3.52
2.74
5.12
4.72
3.00
3.58
3.87
4.12
2.79
3.51
3.74
2.57
CB
Emphys
CB
CB
CB
Emphys
CB
CB
Emphys
CB
Emphys
Emphys
COPD
CB
COPD
125
177
143
137
144
151
168
142
154
139
153
157
149
147
152
37
53
44
41
44
45
51
43
46
40
47
46
45
42
47
160
235
189
312
385
260
308
254
235
133
248
218
335
257
248
4.13
2.64
3.46
2.98
2.57
3.12
3.54
3.21
2.20
1.79
2.29
3.47
4.24
4.59
4.37
Diagnosis
Hb
Pit
Fibrinogen
g·[·l
CB: chronic bronchitis; Emphys: emphysema; COPD: chronic obstructive pulmonary disease; M: male; F: female; Hb:
haemoglobin; Ht; haematocrit; Pit: platelets.
Table 2. - Baseline characteristics of the two different
groups (mean values:so)
Normal pulmonary High pulmonary
artery pressure
artery pressure
No.
PAP mrnHg
PWP mrnHg
Cl l·min"1·m2
PVR
dyn·s·cm·'
Po2 mrnHg
Paco2 mrnHg
pH
FVC l
FEV1 l
FEV/FVC%
MMFR50 t-s·1
14
14:4
6:2
3:1
113:52
15
33:11
9:5
3:1
321:125
70:16
40:6
7.39:0.03
2.62:0.35
1.33:0.33
50:8
0.83:0.36
66:11
45:8
7.40:0.05
2.17:0.88
0.76:0.21
42:11
0.63:0.35
PAP: pulmonary artery pressure; PWP: pulmonary wedge
pressure; Cl: cardiac index; PVR: pulmonary vascular resistance;
Po2: oxyge.n tension; Pco1: carbon dioxide tension; FVC: forced
vital capacity; FEV1: forced expiratory volume in one second;
FEV/FVC: percentageofFVC expiredinthefirstsecond; MMFR:
maximum mid-expiratory flow rate .
Right ventricular, pulmonary artery and pulmonary
wedge pressure were obtained through a Swan-Ganz
catheter and recorded by Hewlett-Packard polygraphic
recorder. Pressures were recorded as the mean of ten
cardiac cycles to minimize respiratory variations.
Blood gases were analysed by an ABL-1 gas analyser
(Radiometer, Copenhagen, DK). Cardiac output was
measured using Fick principle. Pulmonary vascular
resistance was calculated according to standard formulae: pulmonary vascular resistance = (mean pulmonary
artery pressure - pulmonary wedge pressure/cardiac
output) x 80.
Platelet aggregates were measured by the method of
Wu and HoAK [7]. Blood samples were obtained through
the Swan-Ganz catheter from pulmonary artery,
pulmonary capillaries and right ventricle and by a 19G
siliconized needle from an antecubital vein. The first 2
ml of blood were discarded and 1 ml was collected within
5 s into two different 2.5 ml polypropylene syringes
containing, respectively, 1 ml of edetic acid (EDTA)
and 1 ml EDTA-formalin buffer. The sequence of
collecting the two samples was at random. Platelets in
the supernatant obtained by centrifugation at 180 x g for
8 min were then counted by an electronic platelet counter
149
LOCAL PLATELET ACilVATION IN PH DUE TO COPD
(PL 100, TOA, Kobe, Japan). The results were expressed
as a ratio of the platelet count in the buffer-EDTAformalin solution divided by the platelet count in the
buffer EDTA solution.
Beta-TG was assayed in platelet poor plasma according
to Lunu.M et al. [8] using a commercial kit (Beta-TG
RIA - Amersham, UK). Blood, from the same sites as
for platelet aggregates, was collected into cold
polypropylene syringes and immediately transferred into
plastic tubes containing prostaglandin E1 (PGE1) and
theophylline, precooled in a crushed ice water bath, and
then centrifuged at 1,500-2,000 x gat 4°C for 45 min to
obtain platelet poor plasma. Intra- and interassay
variation coefficient were 5.7 and 6.5%, respectively.
Statistical analysis
Results are expressed as mean±standard deviation.
Statistical analysis was performed using one and two
way analysis of variance, Student's t-test for unpaired
data and linear regression.
Results
The baseline pulmonary haemodynamics, levels of
gases in blood and ventilatory tests of patients are reported
in table 2.
In both groups of patients suffering from COPD (with
normal pulmonary artery pressure (group I) and with high
pulmonary artery pressure (group 11), respectively),
platelet aggregates and beta-TG levels in peripheral
venous blood were significantly higher than in control
healthy adults (platelet aggregate ratio: 0.83±0.09 in group
I and 0.80±0.10 in group 11, respectively, vs 0.99±0.02
in control subjects, F=14.9, p<O.OOI; beta-TG: 9.32±2.32
ng·ml·1 in group I and 13.42±4.06 ng·ml·1 in group 11,
respectively, vs 4.35±0.63 ng·mi"l in control subjects
F=38.4, p<0.001).
In COPD patients with normal pulmonary pressure the
numb_er of platelet aggregates found in the three different
sampling sites of the pulmonary circulation was no
different from that in peripheral venous blood (platelet
aggregate ratio: 0.83±0.09 in peripheral vein, 0.85±0.10
in right ventricle, 0.87±0.07 in pulmonary artery and
0.89±0.12 in samples drawn from arteriolo-capillary bed,
F=0.2). On the contrary, in the COPD patients with
PAR
0.9
0.8
0.7
0.6
0.5
perlph. vein
right ventr.
pulm. art
arterlolo-caplll.
Fig. 1. - Platelet aggregates (expressed as platelet aggregate ratio (PAR))
in blood from different sampling sites in COPD patients with normal
pulmonary artery pressure (NPAP) and in patients with pulmonary
hypertension. COPD: chronic obstructive pulmonary disease; periph:
peripheral; ventr.: ventricle; art.: artery; pulm.: pulmonary; capill.: cap·
illaries. • : COPD NPAP; ~:pulmonary hypertension.
ICII
80
50
E
E
40
30
f
20
10
0
:1:
•
o.o
0.2
0.4
0.8
19
••
•
fi
••
•
0.8
1.0
!
~
800
500
400
~
E 4030
f 20
•r = 0.58
300
•
200
100
0
0.0
0.2
·'0.8•
0.8
0.4
PAR
80
50
•
•
• •
1.0
PAR
•• •
10
0 +---~--~--------~-------,
0
10
20
30
19
fi
!
~
800
800
•
•
400
300
200
100
0
•
•
r • 0.50
0
10
20
30
JI·TG ng·mr1
Fig. 2. - Relationship among pulmonary artery pressure (PAP), pulmonary vascular resistance (PVR), platelet aggregate ratio (PAR) and beta-TG plasma
levels. Beta-TG: beta-thromboglobulin.
150
C. ROSTAGNO ET AL.
pulmonary hypertension a significant increase of platelet
aggregates was found in arteriolo-capillary blood (platelet aggregate ratio: 0.80±0.10 in peripheral vein, 0.81±0.17
in right ventricle, 0.77±0.20 in pulmonary artery and
0.60±0.17 in arteriolo-capillary blood, F=7.98, p<0.001)
(fig. 1). A negative linear correlation was found between
platelet aggregate ratio in arteriolo-capillary blood and
pulmonary vascular resistance (r=0.57, p<0.001). On the
contrary, no correlation was found between platelet
aggregate ratio in arteriolo-capillary blood and either mean
pulmonary artery pressure (r=0.33, Ns) or arterial oxygen
partial pressure (r=0.17, Ns) (fig. 2). Beta-TG plasma
levels were higher both in peripheral venous blood and
in the pulmonary vascular bed in patients with
pulmonary hypertension in comparison to normotensive
patients (9.32±2.32 ng·ml· 1 in group I vs 13.42±4.06
ng·m!'l in group 11 in peripheral vein, p<0.005; 9.25±3.08
ng·ml· 1 in group I vs 16.50±4.20 ng·ml· 1 in group 11 in
right ventricle, p<O.OOl; 10.45±3.25 in group I vs
15.02±2.38 ng·ml·1 in pulmonary artery, p<O.OOl). BetaTG levels, as the platelet aggregates, reached the highest
values in arteriolo-capillary blood (11.02±4.02 ng·ml·1 in
patients with normal pulmonary artery pressure vs
18.10±1.79 ng·ml· 1 in patients with pulmonary
hypertension) (fig. 3). Beta-TG plasma levels in arteriolocapillary blood appeared to be mildly related both to
mean pulmonary artery pressure (r=0.50, p<0.01) and
pulmonary vascular resistance (r=0.50, p<0.01), while
no correlation was found between beta-TG levels and
arterial oxygen pressure (r=0.30, Ns) (fig. 2).
Beta-TG ng·ml·'
perlph. vein
right ventr.
pulm. art
arterlolo-caplll.
Fig. 3. - Beta-TG plasma levels in blood from different sampling sites in
chronic obstructive pulmonary disease (COPD) patients with normal
pulmonary artery pressure (NPAP) and in patients with pulmonary
hypertension. : COPD NPAP; ~ :pulmonary hypertension.
Beta-TG: beta-thromboglobulin. For meanings of other abbreviations see
legend to figure 1.
Discussion
These results indicate that a significant platelet activation occurs in hypoxaemic patients suffering from
COPD as indicated by the increased number of platelet
aggregates and increased beta-TG concentrations in
pulmonary blood samples.
A potential bias in our study is the fact that investigation of platelet function by sampling through cardiac
catheters may be misleading in that to some degree platelet
activation could result from interaction of blood with the
catheter itself [9]. Previous investigations dealing with
the reliability of platelet function tests performed in samples collected through different cardiac catheters gave
opposite results [10, 11]. Preliminary experiments
performed by our group to evaluate the reliability of blood
sampling through catheters [12] did not show any
significant difference in beta-TG, thromboxane B2 (TxB2)
6-keto-PGF,~a and PGE2 levels in samples drawn from
an antecubital vein by a 19G siliconized needle and
through a catheter positioned in the brachial vein.
Moreover, an artifactual increase of beta-TG and platelet aggregates is conceivably poorly relevant in relation
to the aim of the present study. This, in fact, was
directed not to the evaluation of beta-TG and platelet
aggregates absolute values but to demonstrate differences
between two different kinds of patients. It must be
acknowledged, however, that in conditions of enhanced
platelet reactivity, as in patients with pulmonary
hypertension, platelet ex vivo stimulation induced by
catheter could be somewhat amplified.
Systemic hypoxaemia and acidosis [13] are known to
cause platelet activation. A shortened platelet survival
and platelet regeneration time were previously reported
in hypoxaemic patients with COPD [4, 5]. Oxygen therapy
resulted only in a partial reversal of these findings [14].
In our patients oxygen desaturation and decreased pH
may play some role in platelet activation, as suggested
by the higher concentration of beta-TG and number of
platelet aggregates in venous samples from the two groups
of hypoxaemic patients in comparison to healthy subjects.
However, in spite of the similar degree of hypoxaemia
and acidosis, only patients with pulmonary hypertension
showed evidence of platelet activation in samples
withdrawn from the arteriolo-capillary bed.
Platelet activation was previously reported in patients
with primary pulmonary hypertension [15] and we found
increased levels of beta-TG and increased platelet aggregates in samples withdrawn from the arteriolo-capillary
bed in four patients with primary pulmonary hypertension (unpublished data). These findings suggest that in
patients with pulmonary hypertension platelet activation
occurs as a consequence of the increased pulmonary artery
pressure itself rather than being a causal mechanism.
However, the release of vasoconstrictor agents, such as
thromboxane (Tx~ or serotonin, from activated platelets can contribute to a further increase in pulmonary
vascular resistance in these patients. At present the
mechanism(s) leading to local platelet activation are
unknown. In conditions of increased systemic blood
pressure an enhanced platelet activation has been reported
due both to the increased shear stress forces and to the
changes in the arteria-arteriolar endothelium [16-18].
Thus, the enhanced platelet activation observed in COPD
patients with pulmonary hypertension is likely to be related to an increased shear stress in the pulmonary
vascular bed, leading to modifications of the
antithrombotic properties of the endothelium and to
altered vessel wall-platelet interactions.
LOCAL PLATELET ACTIVATION IN PH DUE TO COPD
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Preuve d'une activation locale des plaquettes dans les vaisseaux
pulmonaires chez les patients atteints d'hypertension pulmonaire
secondaire a une maladie pulmonaire obstructive chronique.
C. Rostagno, D. Prisco, M. Boddi, L. Poggesi.
REsUME: Afin de rechercher Ies relations entre ]'activation
plaquettaire locale dans les vaisseaux pulmonaires et la pression
arterielle pulmonaire, nous avons evalue Ies agregats des
plaquettes circulantes et le taux de beta thromboglobuline
plasmatique (beta-TG) dans le sang veineux peripherique et a
differents niveaux de la circulation pulmonaire (ventricule droit,
artee pulmonaire et capillaires pulmonaires) chez 29 patients
atteints de broncho-pneumopathie chronique obstructive. Quinze
patients souffraient d'hypertension pulmonaire et 14 etaient
normotendus. Chez les patients normotendus, il n'y avait pas
de difference dans I'agregation plaquettaire on la concentration
de beta-TG aux differents niveaux de prelevement. Au contraire,
chez Ies patients atteints d'hypertension pulmonaire nous avons
trouve une augmentation significative des agregats plaquettaires
et de la concentration de beta thromboglobuline dans le sang de
la circulation capillaire pulmonaire. Nous avons trouve aussi
une correl!ition significative entre l'agregation plaquettaire et la
tension pulmonaire moyenne et Ies resistances vasculaires
pulmonaires. Ces resultats indiquent qu'il y a une activation
plaquettaire locale dans la circulation pulmonaire chez Ies patients avec hypertension pulmonaire consecutive a la
bronchopneumopathie obstructive. Cette activation, a notre avis,
peut contribuer au maintien des hautes resistances vasculaires
pulmonaires.
Eur Respir J., 1991, 4, 147-151.